This invention relates to diagnostic methods based upon a polymorphism in the Tumor Necrosis Factor beta gene (TNFPxcex2; also known as Lymphotoxin alpha), more specifically, an adenine (xe2x80x9cAxe2x80x9d) at the +250 site rather than the usual guanosine (xe2x80x9cGxe2x80x9d). More specifically, this invention relates to a method for diagnosis of Community Acquired Pneumonia (CAP) and diagnosing pre-disposition or susceptibility to CAP, by screening for the presence of this polymorphism, either alone or in combination with a polymorphism at the 308 locus. The invention also relates to compositions for screening for the polymorphism and improved treatment choices for patients diagnosed with CAP or as being susceptible to CAP by the method of the present invention. Further, this invention relates to identifying and diagnosing pre-disposition for septic shock in patients with CAP.
Pneumonia is a common clinical entity, particularly among the elderly. CAP is a major health problem world-wide. In the United States, CAP is the leading cause of death due to infection and the sixth most common cause of death overall. Clinically, CAP exhibits an enormous variety in the severity of presentation, from fulminant septic shock at one end of the spectrum to almost asymptomatic disease at the other. A thorough understanding of the epidemiology and microbiology of Community Acquired Pneumonia (CAP) is essential for appropriate diagnosis and management. Although the microbiology of CAP has remained relatively stable over the last decade, there is new information on the incidence of atypical pathogens, particularly in patients not admitted to hospital, and new information on the incidence of pathogens in cases of CAP and in CAP in the elderly. Recent studies have provided new data on risk-factors for mortality in CAP, which can assist the clinician in making decisions about the need for hospital admission. The emergence of antimicrobial resistance in Streptococcus pneumonia, the organism responsible for most cases of CAP, has greatly affected the approach to therapy, especially in those patients who are treated empirically. Guidelines for the therapy of CAP have been published by the American Thoracic Society, the British Thoracic Society, and, most recently, the Infectious Diseases Society of America and others. These guidelines differ in their emphasis on empirical versus pathogenic-specific management.
CAP remains a significant health problem and patients continue to die despite receiving appropriate antibiotic therapy. Modification of the host immune response, both anti- and pro-inflammatory approaches, has yet to live up to the promise of improved outcome. Despite this, there is significant reason for optimism. Some immunomodulatory therapies clearly have efficacy in some patients. As the understanding of the immune response to pneumonia improves, the ability to tailor specific therapies for individual patients will also improve, hopefully avoiding the deleterious effects that have so far prevented the development of an effective immune-based therapy. The possibility of delivering cytokines directly to the lung, is a particularly promising way of achieving the desired pulmonary effect without systemic side effects. Corticosteroids are currently unique in that they have a proven role in the therapy of pneumonia due to P. carinii. The development of pathogen specific therapies, such as INF for L. pneumophila, based on an improved understanding of host-pathogen interactions, are awaited.
Once respiratory failure has ensued, supportive measures such as patient positioning and differential lung ventilation can improve oxygenation at no additional risk in some patients, particularly those with severe unilateral pneumonia. In facilities where ECMO is available it may be beneficial in selected patients when all other means of providing respiratory support have failed. The role of inhaled NO and partial liquid ventilation is also currently unclear and awaiting further study.
The past 20 years has seen an explosion in knowledge of human immunology and exploration of the therapeutic possibilities is just beginning. The next 10 years promises to finally provide a significant advance in the therapy of pneumonia, the first substantial gain since penicillin.
In light of the prevalence of CAP and the evolution of resistance in the most common bacterial CAP pathogen, physicians advise obtaining specimens for culture of CAP pathogens and analysis of patterns of susceptibility, especially of S. pneumonia, in their communities; using antibiotics appropriately and prudently, according to prevailing susceptibilities when empirical treatment is called for; and immunizing susceptible patients with pneumococcal and influenza vaccines. This is because the mortality of patients with CAP approaches or may exceed 20%, compared to less than 1% for patients with non-severe CAP (Fine et al. New Engl. J. Med. 1997;336:243-250, British Thoracic Society, Q. J. Med 1987;239:192-220, Niederman et al. Am. Rev. Resp. Dis. 1993;148:1418-1426). In such cases an ability to improve accuracy of diagnosis of, or predisposition or susceptibility to CAP, would be of distinct advantage and may lead to improved outcomes and lower medical costs for such patients.
TNF acts on many healthy cells in addition to cancer cells. It is important in regulating immune and inflammatory response and plays a large role in septic shock. It is released by a variety of cells including red and white blood cells, cells that line blood vessels, nervous system cells, muscle cells, bone cells, and some tumor cells. Although it was first observed to kill certain tumor cells (sarcoma cells), TNF has been found to help some tumors grow. In addition, TNF can be very toxic to normal cells. Early experiments found that administering TNF caused fever and loss of appetite. TNF also has been shown to affect the metabolism of many cell types, causing them to need more oxygen. It has been found to play a role in many autoimmune diseases, such as rheumatoid arthritis and myasthenia gravis. Certain viral and bacterial infections can cause healthy cells to produce elevated levels of TNF.
It is a surprising feature of the present invention to be able to diagnose the presence of CAP and the predisposition or susceptibility to CAP by the method of the present invention.
It is an object of the present invention to improve diagnosis of CAP. It is a particular object of the invention to provide a method of diagnosis of predisposition or susceptibility to CAP. A further object is to provide, following such diagnosis, a method of identifying patients for alternative management of CAP before the disease becomes significantly established. Thus, the invention also relates to compositions for screening for the TNFxcex2 polymorphism at the +250 site and improved treatment choices for patients diagnosed with CAP or as being susceptible to CAP by the method of the present invention. A further object of the present invention is to identify and diagnose CAP patients at an increased risk for CAP associated septic shock.
Other preferred embodiments of the present invention will be apparent to one of ordinary skill in light of the following description of the invention and of the claims.
In a first aspect, the invention provides a method of diagnosing a disease associated with a genetic polymorphism (adenine (xe2x80x9cAxe2x80x9d) at the +250 site rather than the usual guanosine (xe2x80x9cGxe2x80x9d)) in a TNFxcex2 gene in an animal predisposed or susceptible to said disease, said method comprising determining the genotype of TNFxcex2 in said animal. The first aspect of the invention further provides a method of identifying an animal predisposed or susceptible to a disease associated with a genetic polymorphism in a TNFxcex2 gene, said method comprising determining the genotype of said TNFxcex2 gene in said animal. In an embodiment of the invention, the method of diagnosis is to screen for an individual at risk of a condition or disease such as CAP correlated with a TNFxcex2 gene polymorphism (xe2x80x9cAxe2x80x9d) at the +250 site.
The invention is based upon a correlation between polymorphisms in the TNFxcex2 gene, specifically at position +250, and predisposition or susceptibility to CAP. The invention is of advantage in that by screening for the presence of the polymorphism it is possible to identify individuals likely to have a genetic predisposition or susceptibility to the disease. It may also result in substantially different management, both prevention and treatment, if pneumonia occurs, with subsequent substantial improvement in mortality and morbidity from CAP.
In an embodiment of the invention, diagnosis is carried out by determining whether a TNFxcex2 gene contains a polymorphism xe2x80x9cAxe2x80x9d at position +250. Possessing a fragment that contains the xe2x80x9cAxe2x80x9d at that +250 site correlates with increased risk of predisposition or susceptibility to CAP. As a human genome contains two TNFxcex2 genes, one on each of a pair of chromosomes, an individual can accordingly be found to be homozygous or heterozygous for the risk polymorphism, or to lack the risk polymorphism.
Genotypic and allelic frequencies of this invention are readily determined by a number of methods known to those skilled in the art. Methods used in the present invention are shown in Example 1 below and include using PCR amplification and restriction enzyme digestion. The method conveniently comprises amplifying a fragment of a TNFxcex2 gene to produce copies and determining whether copies of the fragment contain the polymorphism.
Another suitable technique is to amplify the fragment using PCR techniques, producing copies of a fragment that is at least 500 base pairs in length, preferably at least 600 base pairs in length. It is preferred that the PCR primers are selected so as to amplify a region of the gene that is about 740 base pairs in length. PCR techniques are well known in the art and it would be within the ambit of a person of ordinary skill in this art to identify primers for amplifying a suitable section of the applicable exon of the TNFxcex2 gene. PCR techniques are described for example in EP-A-0200362 and EP-A-0201 184. In a further embodiment of the invention, the diagnostic method comprises analysis of the TNFxcex2 gene using single strand conformational polymorphism (SSCP) mapping to determine whether the TNFxcex2 gene is the risk or the non-risk form.
In preferred embodiments, the method comprises screening a TNFxcex2 gene, and this screening is conveniently carried out by any one of a number of suitable techniques that are known in the art, and may be conveniently selected from amplification of a nucleic acid sequence located within the TNFxcex2 gene, Southern blotting of regions of the gene and single strand conformational polymorphism mapping of regions within the gene or as described in Example 1 below. The genotype in that region is also optionally determined using hybridization probes adapted selectively to hybridize with the particular polymorphism of the TNFxcex2 gene at the +250 location which is associated with predisposition or susceptibility to disease. A probe used for hybridization detection methods must be in some way labeled so as to enable detection of successfully hybridization events. This is optionally achieved by in vitro methods such as nick-translation, replacing nucleotides in the probe by radioactively labeled nucleotides, or by random primer extension, in which non-labeled molecules act as a template for the synthesis of labeled copies. Other standard method of labeling probes so as to detect hybridization are known to those skilled in this art.
According to a second aspect of the invention, there is provided a method of diagnosis and therapy comprising diagnosing CAP and diagnosing predisposition or susceptibility to CAP according to the method of the invention and treating an individual having such CAP or a predisposition or susceptibility to CAP thereby treating to prevent or lessen CAP.
Known therapies for CAP can be effective in halting advancement of the disease, or at least slowing the advancement. TNFxcex2+250 gene analysis may also lead to more appropriate placement of patients into intensive care/critical care units, an important factor in optimizing survival from CAP. It is thus an advantage of the invention that early diagnosis of CAP is improved, so that preventative therapy can be started as soon as possible, optimizing any interventions potential (such as immunomodulatory therapy) for affecting outcome. As alternative diagnostic methods improve and are developed, so the invention can add to the total knowledge of the risk of developing CAP of an individual. The decision of a physician on how and whether to initiate therapy in anticipation of the disease can be taken with increased confidence.
A variety of suitable treatments of CAP are described in the art. See e.g., Hirani and MacFarlane Thorax 1997;52:17-21, Pachon J et al. Am. Rev. Resp. Dis. 1990;142:369-373, Ruiz M et al. Am. J. Respir. Crit. Care. Med. 1999;160:923-929, Leeper and Torres Clin. Chest. Med. 1995;16:155-171. Other treatments are known to persons of skill in the art.
Another aspect of the invention provides a composition for use in diagnosing a disease associated with a genetic polymorphism in a TNFxcex2 gene in an animal predisposed or susceptible to said disease, said composition comprising one or more primer nucleic acid molecules adapted to amplify a portion of a TNFxcex2 gene selected from a portion of the gene around the +250 location.
Another aspect of the invention also provides a composition for use in identifying an animal predisposed or susceptible to a disease associated with a genetic polymorphism in a TNFxcex2 gene, said compositions comprising one or more primer nucleic acid molecules adapted to amplify a portion of the TNFxcex2 gene selected from a portion of the gene around the +250 location.
The composition of this aspect of the invention may comprise a nucleic acid molecule capable of identifying the +250 polymorphism in said TNFxcex2 gene, said polymorphism being indicative of a risk genotype in said animal.
A further embodiment of the invention provides a composition for diagnosis of CAP or predisposition or susceptibility to CAP, comprising means for determining genotype of a TNFxcex2 gene of an individual, for example whether an individual is homozygous or heterozygous for polymorphic variants of a TNFxcex2 gene at the +250 location such as the method provided in the Example 1 herein.
In an embodiment of the invention, a diagnostic composition comprises PCR primers adapted to amplify a DNA sequence within and around the TNF +250 polymorph location, wherein alternative versions of the gene are distinguished one from another.
In a further aspect of the invention there is provided a diagnostic kit comprising a diagnostic composition as described above and an indicator composition for indicating how possessing a polymorphic version of a TNFxcex2 gene correlates with the presence of CAP or redisposition or susceptibility to CAP.
Diagnostic kits are typically accompanied by or comprise a chart or other visual aid for assistance in interpreting the results obtained using the kit. Suitable indicator compositions for use in the diagnostic kit of the invention include a leaflet or other visual reminder that possessing the risk polymorphism version of a TNFxcex2 gene correlates with CAP or the increased risk of predisposition or susceptibility to CAP.
In a still further aspect of the invention there is provided use, in the manufacture of means for diagnosing whether an individual has a predisposition or susceptibility to CAP, of PCR primers adapted to amplify a region around +250 in the TNFxcex2 gene. Alternative versions of the gene are typically distinguished one from another by means known to those skilled in the art.
Multiple techniques exist and are known to one skilled in the art in the manufacture of means for diagnosing whether an individual has CAP or a predisposition or susceptibility to CAP, of PCR primers adapted to amplify a region around 250 in the TNFxcex2 gene. Restriction analysis can be employed, where the enzyme cuts if a xe2x80x9cGxe2x80x9d is present, but not if an xe2x80x9cAxe2x80x9d is present, and when run on a, e.g., 1% agarose gel, the different fragments migrate differently based upon size.
According to the invention, an individual who is homozygous for a risk polymorphism, that is to say homozygous for a version of 250 (xe2x80x9cAAxe2x80x9d), is classified as being at highest risk of CAP or predisposition or susceptibility to CAP. An individual being heterozygous (xe2x80x9cGAxe2x80x9d) is classified as having moderate risk of CAP or predisposition or susceptibility to CAP.
Optionally, the assessment of an individual""s risk factor according to any aspect of the invention is calculated by determining the genotype of a TNFxcex2 gene polymorphism and combining the result with analysis of other known genetic, physiological, dietary, clinical, or other indications known to those of skill in the art. The invention in this way provides further information on which measurement of an individual""s risk can be based.
It is possible that CAP polymorphisms are not the disease causing genes. Nevertheless, the observed correlation is of use in diagnosis of CAP and of risk of predisposition or susceptibility to CAP.
In another embodiment of the invention, the results of the genotyping done herein are used, along with other diagnostics measures and disease parameters, by treatment providers to determine the best course of treatment for the patient having CAP or having been determined as susceptible to CAP by the methods of this invention.
In another aspect of the invention, a method of identification and diagnosis of patients with CAP at an increased risk of septic shock is provided. A significant association between the LTxcex1+250 genotype and the risk of septic shock in patients with CAP has been identified. In addition, analysis of LTxcex1+250: TNFxcex1xe2x88x92308 haplotypes strongly suggests that the LTxcex1+250 locus is not the causative polymorphism. This finding sheds some light on the relative importance of the LTxcex1+250 and TNFxcex1xe2x88x92308 loci with respect to CAP.
Haplotype analysis has shown that the lowest risk of septic shock is in patients with two 250G:308G haplotypes. It is believed that the TNFxcex1xe2x88x92308 locus does exert an influence, but is largely masked by the much greater prevalence of the A allele at the LTxcex1+250 locus in the population. This shows the important role that differences in local allelic frequencies play in evaluating the significance of gene.
These findings have important implications for understanding the inflammatory response to severe infections. In an effort to identify patients at high risk for subsequent septic shock, the systemic inflammatory response syndrome (SIRS) criteria were developed. SIRS is defined as having at least two of the following four conditions: (1) oral temperature of  greater than 38xc2x0 C. or  less than 36xc2x0 C.; (2) respiratory rate of  greater than 20 breaths/minute or Paco2 or  less than 32 count torr; (3) heart rate of  greater than 90 beats/minute; (4) leukocyte count of  greater than 12,000/xcexcl  or  less than 4,000/xcexcl  or  greater than 10% bands. Hypoxemia was included as an organ dysfunction and is one of the two most common organ dysfunctions in most studies of SIRS. Hypoxemia (and other organ dysfunctions in SIRS) is assumed to occur by the same mechanism and reflect the same pro-inflammatory state as septic shock. This unexpected finding that respiratory failure is not associated with a TNF hypersecretor genotype, with a trend to greater respiratory failure with a TNF hyposecretor genotype (LTxcex1+250 GG) is not consistent with this understanding of SIRS.
It is believed that LTxcex1+250 is not directly causative, but is a marker for the xe2x80x98realxe2x80x99 polymorphism which is located within the 250A:308G haplotype. The A:G haplotype is considered a risk version of the gene. This has important implications not only for understanding the molecular basis for susceptibility to septic shock, but also for other studies showing association between disease states and LTxcex1+250 genotype. It is further believed that both polymorphisms may be causative, but an A allele at one locus interferes with the mechanism leading to increased TNFxcex1 production, (such as a conformation change that affects the binding of transcription activating factors) with the A allele at the other locus.
Clearly non-genetic factors such as the length of time to initial therapy and adequacy of therapy also play important roles in the ultimate development of the clinical presentation, including the development of septic shock. These studies did not control for the length of time from onset of symptoms to presentation. However, this data supports the hypothesis that genetic polymorphisms play a critical role in the variable presentation of CAP.
Differences in the virulence of pathogens also has an impact on outcome of CAP. These experiments did not analyze the influence of genotype on CAP for individual pathogens due to the low number of cases with a definite microbiological diagnosis. The problem of the low sensitivity of traditional culture techniques in CAP is well known, and the diagnostic rate observed is not inconsistent with studies of the yield of cultures in patients with CAP. However, the pattern in patients with proven or suspected pneumococcal CAP parallels that of the entire cohort, a finding which is not surprising given that the pneumococcus is the most common cause of CAP.
Septic shock is associated with the TNFxcex1 high secretor genotype (AA) at the LTxcex1+250 locus while type I respiratory failure in the absence of septic shock is associated with the low secretor (GG) genotype. The TNFxcex1xe2x88x92308 polymorphism appears to have the similar associations, but a lesser influence in this cohort due to the comparative rarity of the TNFxcex1xe2x88x92308A allele and linkage disequilibrium with the LTxcex1+250 polymorphism. The finding that these two presentations of CAP have opposite associations with respect to TNFxcex1 secretion raises significant concern regarding the validity of the SIRS definition as an inclusion criteria for anti-inflammatory sepsis trials.